Proteins destined to be secreted or expressed on the cell surface are synthesized in the endoplasmic reticulum (ER) where they acquire their biologically active structure. As these proteins enter the ER, they encounter a variety of molecular chaperones, including two Hsp70s and their DnaJ co-factors, which bind directly to the nascent proteins and either aid their folding and assembly into multi-subunit complexes or target them for degradation if they fail to mature properly. How the ER sets the threshold for proteins to be transported along the secretory pathway, retained in the ER for further folding attempts, or targeted for degradation is an essential biological question that is not well understood. We hypothesize that recognition of specific sequences by the various chaperones of the ER is likely to provide a critical aspect of this quality control discrimination, although te in vivo specificity of ER chaperones has never been determined. To address this important gap of knowledge, we have developed an in vivo expression system/library composed of a series of 25-amino acid, overlapping peptides covering critical regions for quality control of an immunoglobulin ??heavy chain and a complete non-secreted Ig light chain, both of which bind directly to the ER Hsp70 chaperones and their co-factors. The peptides are expressed in the ER of mammalian cells via their fusion to an ER-targeted, well-folded Ig domain, which importantly does not bind any of these chaperones. Characterization of these libraries reveals that BiP and ERdj3 bind multiple peptides throughout both clients, whereas Grp170, ERdj4, and ERdj5 binding sites are much rarer and more restricted in their locations. Aim 1 will define binding site for BiP and Grp170 and use this information to determine the biological consequences of inhibiting substrate interactions with these two chaperones, as well as the functional relationship of the two ER Hsp70s to each other. In Aim 2, after delineating binding sequences on our model proteins for the three ERdj co-factors, studies are proposed to understand the mechanistic interactions occurring between the ERdj proteins and with the Hsp70 chaperones that lead to disparate outcomes for clients. Proteins that fail ER quality control are extracted from the ER and degraded in the cytoplasm by the 26S proteasome; processes that are dependent on their targeting to the retrotranslocon and ubiquitination. Using our series of peptide constructs, in Aim 3 we will develop an in vitro ubiquitination assay for mass spectrometric analyses base on our peptide libraries to identify amino acids that are modified for the purpose of ER quality control. comparative approach will be used to elucidate components of the ERAD machinery for non-glycosylated proteins, which are currently poorly defined. The data obtained from the combination of these studies will provide critical insights into the molecular determinants and mechanisms that underlie ER quality control.